Abstract

Purpose: To describe the clinical and
genetic findings in one Chinese family with juvenile-onset open angle
glaucoma (JOAG).

Methods: One family was examined
clinically and a follow-up took place 5 years later. After informed
consent was obtained, genomic DNA was extracted from the venous blood
of all participants. Linkage analysis was performed with three
microsatellite markers around the MYOC gene (D1S196, D1S2815,
and D1S218) in the family. Mutation screening of all coding exons of MYOC
was performed by direct sequencing of PCR-amplified DNA fragments and
restriction fragment length polymorphism (RFLP) analysis.
Bioinformatics analysis by the Garnier-Osguthorpe-Robson (GOR) method
predicted the effects of variants detected on secondary structures of
the MYOC protein.

Results: Clinical examination and
pedigree analysis revealed a three- generation family with seven
members diagnosed with JOAG, three with ocular hypertension, and five
normal individuals. Through genotyping, the pedigree showed a linkage
to the MYOC on chromosome 1q24–25. Mutation screening of MYOC
in this family revealed an A→T transition at position 1348 (p. N450Y)
of the cDNA sequence. This missense mutation co-segregated with the
disease phenotype of the family, but was not found in 100 normal
controls. Secondary structure prediction of the p.N450Y by the GOR
method revealed the replacement of a coil with a β sheet at the amino
acid 447.

Conclusions: Early onset JOAG, with
incomplete penetrance, is consistent with a novel mutation in MYOC.
The finding provides pre-symptomatic molecular diagnosis for the
members of this family and is useful for further genetic consultation.

Primary open-angle glaucoma (POAG;OMIM 137760) is one of
the leading causes of blindness in the world [1]. It is a
neurodegenerative disorder characterized by progressive excavation of
the optic discs due to loss of retinal ganglion cells. It is usually
associated with elevation of intraocular pressure (IOP) [2]. Based upon the age
of diagnosis, primary open-angle glaucoma can be sub-classified to
either juvenile-onset primary open-angle glaucoma (JOAG) or adult-onset
primary open-angle glaucoma. JOAG is a relatively rare form of primary
open angle glaucoma that occurs in children and young adults. The exact
age boundary for juvenile-onset varies from one study to the next, but
it usually falls between 35 and 40 years of age [2].

Strong evidence indicates that genetic factors play a role in the
pathogenesis of glaucoma. About 30%–56% of patients with glaucoma or
ocular hypertension (OHT) have a positive family history; first-degree
relatives of POAG patients are seven to ten times more likely to have
POAG, compared with the general population [3,4].
Genetically, most POAG cases follow a complex (non-Mendelian) pattern
of inheritance, which manifests clinically in adulthood (>40 years).
However, juvenile-onset open-angle glaucoma typically shows an
autosomal dominant inheritance [2-4].
To date, three genes, namely myocilin (MYOC), optineurin (OPTN),
and WD repeat-containing protein 36 (WDR36), have been
reportedly linked to POAG [5-10]. MYOC
(OMIM 601652)
was the first gene to be identified as responsible for POAG. Mutations
in MYOC account for over 8% of JOAG and 3%–4% of adult-onset
POAG [11,12].

MYOC, consisting of three exons, encodes 504 amino acid
residues. Myocilin is an acidic protein that contains an NH2-terminal
myosin-like domain and a COOH-terminal olfactomedin-like domain [6]. Almost 80 mutations
have been found in MYOC and about 90% of the mutations are
located in the olfactomedin-like domain encoded by exon3 [6,11-30].

In this study, we describe the clinical findings in a Chinese family
with a novel MYOC mutation.

Patients and DNA sample collection

This study was performed according to the tenets of the Declaration
of Helsinki for research involving human subjects. This study was
approved by the Beijing Tongren Hospital Joint Committee on Clinical
Investigation. After informed consent was obtained, all participants
underwent ophthalmologic examination including bilateral best corrected
visual acuity using E decimal charts, slit-lamp biomicroscopy
inspection of the anterior chamber, intraocular pressure (IOP)
measurement by applanation tonometry (Goldmann), anterior chamber angle
evaluation by gonioscopy (Goldmann), and fundus examination with a
66-diopter VOLK lens. Most members were clinically followed for five
years, from 2004 to 2009. Some individuals underwent Octopus’s
perimeter examination. Diagnosis of POAG was based on the observation
of at least two of the following abnormalities: characteristic
glaucomatous optic disc changes, characteristic glaucomatous visual
field defects, and high intraocular pressure (>21 mmHg) in the
presence of a normal open anterior chamber angle. Characteristic
glaucomatous optic disc changes include vertical cup-disc (c/d) ratio
of 0.7 or more, notching of the neutral rim, and disc hemorrhage.
Subjects were sub-classified JOAG if the diagnosis of POAG was made
before 35 years of age. Individuals with intraocular pressure greater
than 22 mmHg but with no characteristic optic disc damage or
visual field impairment were defined as ocular hypertension. Unaffected
people had IOP in the normal range (≤21 mmHg) and optic nerves
presented normal in appearance.

Linkage analysis

Genotyping and linkage analysis were performed with three
microsatellite markers (D1S196, D1S2185, and D1S218) around the MYOC
gene in the family. The fine mapping primer sequences were obtained
from the GDB Human Genome Database.
LOD scores were calculated for the two markers by two-point linkage
analysis using linkage
package 5.2. We modeled the disease as an autosomal dominant trait with
reduced penetrance. Pedigree and haplotype maps were constructed using Cyrillic version 2.0
software.

Mutation screening of MYOC

Peripheral blood was obtained by venipuncture and genomic DNA was
extracted according to standard protocols. The entire coding region of MYOC
was amplified by polymerase chain reaction (PCR) from genomic DNA.
Primers for three exons and exon-intron boundaries of MYOC were
designed by the Primer3
program. These primer sequences are presented in Table 1.
For direct sequencing, PCR products were purified (Shenneng Bocai PCR
purification kit; Shenneng, Shanghai, China). An automatic fluorescence
DNA sequencer (ABI, Prism 373A; Perkin Elmer, Foster City, CA), used
according to the manufacturer’s instructions, was used to sequence the
purified PCR products in both forward and reverse directions. DNAssist Version 1.0 compared nucleotide
sequences with the published DNA sequence of MYOC (GenBank NM_000261).
For the MYOC gene, cDNA numbering +1 corresponded to the A in
the ATG translation initiation codon of MYOC.

Restriction fragment length polymorphism (RFLP) analysis

To confirm the variations found in the sequencing, restriction
endonuclease HindII (New England Biolabs, Ipswich MA) was used in all
available family members and in 100 normal control subjects. The
reaction was performed in a 10 μl volume containing 9.4 μl
PCR product, 0.1 μl BSA (100 μg/ml), and 0.5 μl enzyme
(10 U/μl). After incubating the reaction overnight at 37 °C, the
entire digest was run on a 1% agarose gel and visualized under
ultraviolet light.

Bioinformatics analysis

Garnier-Osguthorpe-Robson (GOR)
software was used to predict the effect of the mutation on the
secondary structure of MYOC [31].
This method infers the secondary structure of a sequence by calculating
the probability for each of the four structure classes (helix, sheet,
turn, and loop) based on the central residue and its neighbors from the
calculated matrices.

Clinical findings

We have identified a three- generation family diagnosed with JOAG.
The inheritance pattern in this family appeared to be autosomal
dominant (Figure 1).
After clinical examinations and hospital records reviewing, six
individuals of this pedigree were found to have glaucoma in 2004. The
patient in the first generation had not received any treatment and
totally lost her sight before the age of 35. The remaining five
patients underwent trabeculectomies in both eyes. The mean onset age of
these patients was 27.42 years (ranging from 20 to 31 years old), which
was consistent with juvenile glaucoma. All patients experienced
elevated IOP (32–50 mmHg) and most of them presented typical late
stage glaucoma changes in the optic disc and in the visual field (Figure 2A).
In 2004, six members were diagnosed with ocular hypertension (IOPs were
higher than 22 mmHg) but without optic disc or visual field
changes. A five-year follow-up was conducted with fifteen of the
seventeen individuals and their blood samples were collected for
further genetic analysis. At the 5-year follow-up, two ocular
hypertension patients (Figure 1; III:2 and III:7) were newly
diagnosed with glaucoma due to their elevated IOP, enlarged cup/disc
ratio of the optic disc, and early visual field changes in 2009 (Figure 2B) .
Detailed clinical information of the pedigree is summarized in Table 2.

Genotyping results

The family was genotyped with three STRP markers located around the MYOC
gene in the chromosome 1q24–25 region. The marker results for D1S218
and D1S2815 were fully informative for linkage. There was no affected
(glaucomatous patients and ocular hypertension patients) recombinant
for either of the two makers (Figure 1). Two clinical unaffected
individuals (II:12 and III:12), however, were found to be carrying the
affected haplotype. Therefore, the disease penetrance appeared
incomplete in this pedigree. Two-point LOD scores for D1S2815 and
D1S218 with 80% penetrance were 2.40 (θ=0.0) and1.63 (θ=0.0),
respectively.

Mutation analysis

By direct sequencing of three exons of MYOC, we found a
novel base change (A→T) at position 1348 of MYOC cDNA,
replacing asparagine with tyrosine at amino acid 450 residue (Figure 3A).
This heterozygous missense mutation abolished a HindII restriction site
that segregated with all affected members and ocular hypertension
individuals in this Chinese family, but that was not detected in 100
unrelated normal controls. As observed in the genotyping, two clinical
unaffected individuals (II:12 and III:12) carried the mutation as well (Figure 3B).

Prediction of two-dimensional structure

Using the GOR method, the results for secondary structure prediction
suggested that the mutant MYOC450Y replace a coil “C” with a β sheet
“E” at amino acid 447 Figure 4).

This study described a Chinese family with clinically diagnosed
juvenile-onset open angle glaucoma. By screening the MYOC gene,
we identified a novel heterozygous missense mutation p. N450Y in the
pedigree. The mutation p. N450Y co-segregated with all glaucoma
patients and ocular hypertension individuals, but was not detected in
100 normal controls.

MYOC was the first disease-causing gene identified for POAG
and almost 80 mutations have been reported [6,11-30]. Mutations in MYOC
are racial/ethnic specific and some of them have been found only in a
specific region [6,11-30]. So far, 11 MYOC
mutations have been identified in Chinese patients or pedigrees and
seven of them were Chinese specific (Table 3) [19,20,22,23,25,26,28].

The Asn450 residue, located in the olfactomedin-like domain, is
highly conserved in humans, rats, mice, cattle, dogs, and zabrafish (Figure 5).
The results of GOR suggested that p.N450Y lead to a secondary structure
change by replacing a coil structure with a β sheet around the Asn450
residue, which might interfere with the correct folding of the protein.
In a large case control study, another mutation (p. N450D) was also
detected at the Asn450 residue in a sporadic Germany patient [18]. This may imply
that the Asn450 residue is very important for the activity of the
olfactomedin-like domain.

Phenotype and genotype correlation has been well established in some
MYOC mutations [11,12,27]. Patients carrying
the P370L mutation usually developed glaucoma at a very early age, with
high levels of IOP, which responds poorly to medical treatment [12,32,33]; while patients
with the Q368X mutation were diagnosed with glaucoma at a later adult
age and their maximum IOPs were around 30 mmHg, which could be
well controlled by medical therapy [12,34,35]. One American
family carrying the p.D380H MYOC mutation presented with an
intermediate phenotype between juvenile and adult onset glaucoma [36]. In the current
study, the onset age of glaucoma ranged from 20 to 31 years (mean 26
years). The mean highest IOP was 48.57 mmHg (range from 32 to
60 mmHg). One patient totally lost her sight before 35 years of
age. Except for two patients newly diagnosed in 2009, the remaining
five patients responded poorly to medical therapy and required
filtration surgery for long-term IOP control. Five individuals
diagnosed with ocular hypertension in 2004 carried the mutation p.N450Y
and their mean age at diagnosis was 17.8 years. At the 5-year follow
up, two of them presented glaucomatous optic disc change and were newly
diagnosed with glaucoma. The phenotype and genotype correlation study
on seven patients in this pedigree indicated that affected members
carrying the mutation p.N450Y experienced more severe symptoms at an
earlier age.

Incomplete penetrance has been observed in most families with MYOC
mutations and the penetrances are age-dependent and mutation-specific [11,12,27]. The penetrance of
pedigrees carrying p. P370L was 100% at age 30 years [12,32,33], while it was 0
for the pedigrees with Q368X [12,34,35]. In this pedigree,
two clinically healthy individuals and three ocular hypertension
patients were found harboring both mutation p.N450Y and the affected
haplotype. The penetrance of this pedigree was 50% (6/12) at age 30 and
almost 60% (7/12) at age 35 years. More than 80% (10/12) of the
individuals carrying the p.N450Y mutation have developed glaucoma or
ocular hypertension. Interestedly, one of the healthy members (II-12)
was already 39 years old, which was ten years older than the average
onset age of this family; this implied that other unidentified factors
(genetic or environmental) might be associated with the JOAG of this
pedigree. However, whole carriers should undergo ophthalmologic
surveillance at regular intervals for the rest of their lives.

In summary, the report described a novel conserved tyrosine to
asparagine substitution at exon 3 of MYOC associated with an
early-onset and severe juvenile-onset open angle glaucoma pedigree. The
results further expanded the mutation spectrum of MYOC and
characterized the genotype-phenotype correlations of this pedigree.
These results provide pre-symptomatic molecular diagnosis for the
members of the pedigree and are useful for further genetic consultation
with this family.